A major goal of my laboratory is to understand how cytoskeletal polymers establish long-range order in the cytoplasm and help convert a rowdy mob of macromolecules into a living cell. Initially, we focused on actin filament networks that drive migration of eukaryotic cells but, in 2004, our interests expanded to include bacterial polymers (Garner, 2004). Until recently, cytoskeletal polymers like actin filaments and microtubules were thought to be eukaryotic innovations, not present in bacteria. Recent work, however, has identified a number of cytoskeletal systems in bacteria, including actin-like proteins required to maintain cell shape (Jones, 2001); transport cargo through cytoplasm (Moller-Jensen, 2003; Kruse, 2003); and organize intracellular compartments (Komeili, 2006). My laboratory has focused particular attention on the actin-like protein, ParM, which forms dynamic filaments that push cargo to opposite poles of rod-shaped bacteria. The ParM gene is part of a partitioning locus (the par operon) found on many low-copy plasmids (e.g. clinically important R1 and R100 drug-resistance plasmids). To ensure plasmid inheritance the par operon constructs a DNA-segregating spindle from three components: (1) a stretch of centromeric DNA called parC (Dam, 1994); (2) a repressor protein, ParR, that binds the parC locus (van den Ent, 2002); and (3) the actin-like protein ParM. The ParR/parC complex harnesses the energy of ParM polymerization to produce forces that push pairs of plasmids in opposite directions through the cytoplasm (Moller-Jensen, 2002; Moller-Jensen, 2003). We previously characterized the assembly dynamics of ParM filaments (Garner, 2004) and reconstituted ParM-based DNA segregation in vitro using purified components (Garner, 2007). The goal of the present proposal is to understand ParM-mediated plasmid movement in molecular detail. We pursue these studies for several reasons, including: (1) understanding ParM-dependent DNA segregation provides insight into related systems that work to organize prokaryotic cytoplasm. (2) ParM filaments have a simpler architecture than microtubules, making them the best system for studying the molecular basis of dynamic instability. (3) Understanding mechanisms that maintain drug resistance plasmids in bacterial populations can help us deal with the emergence of clinical drug resistance. We will perform quantitative studies at three size scales: (i) single molecule and bulk biochemical studies; (ii) biophysical and microscopical studies of reconstituted ParM spindles; and (iii) cell biological studies of ParM in living cells. PUBLIC HEALTH RELEVANCE: Bacteria are not simply disorganized bags of protein and DNA: many large molecules and complex intracellular structures must be moved through the cytoplasm and properly positioned. In this project we work to understand how large pieces of DNA that confer multi-drug, antibiotic resistance to enteric pathogens are moved to opposite ends of a bacterium so that both they can be inherited by both daughter cells after cell division.